Nitric oxide (NO) is a cell signaling molecule and involved in numerous biological processes, including vasodilatation, neurotransmission, macrophage mediated immunity and carcinogenesis. Nitric oxide (NO) is differentially formed in almost all types of human cells by two constitutive and one inducible form of nitric oxide synthases (NOS). The possible role of NO in fertility has been proposed by many recent publications. Studies with rats have reported that NO has both normal physiological effect on reproduction and inflammation-based infertility. NO has apparent capability to improve sperm viability and motility in both fertile and infertile individuals. However, NO has been implicated as a cause to obstructive azoospermia, one of the common causes of infertility. Like constitutive NOSs, inducible nitric oxide synthase (iNOS) also takes part in protein-protein interaction. Further bolstering the protein-protein interaction of all NOSs, recent research with site- directed mutagenesis has identified critical N-terminal amino acid residues essential for dimerization of human iNOS and its activity. It is noteworthy that the amino acid sequence of three NOSs is different in their N-terminal portions. The use of iNOSs'N-terminus as bait in the yeast two-hybrid system could possibly identify new putative interacting proteins. Since NO has a dual role in human physiology, NOS-isoform specific inhibitors are needed in terms of disease prevention, e.g., hypertension, lung cancer, infertility, etc. This proposal is designed to answer the following question: What other proteins are interacting with iNOS and how does this interaction influence NO production in testis? The proposal's hypothesis is that iNOS interacts with proteins, not yet identified, that modulate its role in testis physiology. The following specific aims will investigate this hypothesis: 1) Isolate and identify genes that encode proteins that bind iNOS in the yeast two-hybrid system;2) Confirm binding specificity between iNOS and putative interacting proteins in vivo using mammalian cell lines;and 3) Characterize the effects of iNOS modulation on NO synthesis affecting testis physiology employing RNA interference study. Consequently, this study will expand current knowledge of the role of iNOS mediated NO synthesis in male fertility, and possibly reveal other pathogenic mechanisms in the reproductive system that may include birth defects due to male germline mutations. PUBLIC HEALTH RELEVANCE: This study will expand current knowledge of the role of iNOS mediated NO synthesis in male fertility, and possibly reveal other pathogenic mechanisms in the reproductive system that may include birth defects due to male germline mutations.